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Toxicological information

Skin irritation / corrosion

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Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2016-04-28 to 2016-10-07
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report Date:
2016

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
(2015)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Specific details on test material used for the study:




STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: Epi-200 SIT kit purchased from MatTek Corporation (Bratislava, Slovakia) containing EpiDerm™ tissues on agarose
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm tissues (surface 0.6 cm2)

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37°C / 60 minutes
- Temperature of post-treatment incubation (if applicable): 37°C / 60 minutes

CONTROL
- Negative Control: 30 µL DPBS (Gibco) was used as negative control per tissue;
- Positive Control: 30 µL of a 5% SLS solution in deionised water (MatTek) was used a positive control per tissue, freshly prepared prior to the start of the experiment.

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: tissues were gently rinsed with DPBS at least 15 times

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/L
- Incubation time: 3 hours
- Spectrophotometer: Versamax Molecular Devices, Softmax Pro, (version 4.7.1.)
- Wavelength: 570 nm

NUMBER OF REPLICATE TISSUES: triplicate

PREDICTION MODEL / DECISION CRITERIA
For the current test, an irritation potential of a test item is determined and classification is recommended if the mean relative tissue viability of three individual tissues is reduced to less than or equal to 50% of the negative control.
Mean tissue viability =50% = irritant (I)
Mean tissue viability >50% = non-irritant (NI)

Acceptability of the Assay
Criterion 1 (negative control): The absolute OD 570 nm of the negative control tissues in the MTT test is an indicator of tissue viability obtained after the shipping and storing procedure and under specific conditions of the assay. Tissue viability is meeting the acceptance criterion if the mean OD570 of the negative control tissues is > 0.8 and =2.8.
Criterion 2 (positive control): An assay is meeting the acceptance criterion if mean relative tissue viability of the positive control is at or below 20%.
Criterion 3 (standard deviation): The SD of 3 identical replicates should be < 18%.
OD values should not be below historically established boundaries.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
30 µL
Duration of treatment / exposure:
60 minutes
Duration of post-treatment incubation (if applicable):
42 hours
Number of replicates:
3

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
% tissue viability
Remarks:
after 60 min
Value:
8.8
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: mean relative absorbance
Other effects / acceptance of results:
- Direct-MTT reduction: test item did not reduce MTT
- Colour interference with MTT: test item did not change colour in the colour interference test
- After treatment with the negative control the absorbance values were well in the required range of the acceptability criterion of mean OD = 0.8 and = 2.8 for the 60 minutes treatment interval, thus assuring the quality of the tissues.
- Treatment with the positive control induced a sufficient decrease in the relative absorbance as compared to the negative control for the 60 minutes treatment interval, and thus assuring the validity of the test system; the mean relative absorbance value of the positive control was 4.3% compared to the negative control.

Applicant's summary and conclusion

Interpretation of results:
Category 2 (irritant) based on GHS criteria
Conclusions:
Under the experimental conditions reported, the test substance is irritant to skin.
Executive summary:

This in vitro study was performed to assess the irritation potential of the test material by means of the Human Skin Model Test.

The test item did not reduce MTT (test for direct MTT reduction), and it did not changen colour when mixed with deionised water (test for colour interference). Also its intrinsic colour was not intensive. Consequently, additional tests with freeze-killed or viable tissues were not necessary. Each three tissues of the human skin model EpiDerm™ were treated with the test item, the negative or the positive control for 60 minutes. 30 µL of the test item were applied to each tissue and spread to match the surface of the tissue. 30 µL of either the negative control (DPBS) or the positive control (5% SLS) were applied to each tissue. After treatment with the negative control the absorbance values were well within the required range of the acceptability criterion of mean OD = 0.8 and = 2.8 for the 60 minutes treatment interval, thus assuring the quality of the tissues.

Treatment with the positive control induced a sufficient decrease in the relative absorbance as compared to the negative control for the 60 minutes treatment interval, and thus assuring the validity of the test system. After treatment with the test item ethyl isovalerate the mean relative absorbance value decreased to 8.8% compared to the relative absorbance value of the negative control. This value is below the threshold for irritancy of = 50%. Therefore, the test item is considered to possess an irritant potential.